Ankyrin (ANK) repeats, identified in thousands of proteins, are composed of pairs of antiparallel alpha- helices that stack on top of each other and form super-helical spiral domains with suggestive spring-like properties, whose primary function is to mediate specific protein-protein interactions. For example, ankyrin-R links the anion exchanger in the erythrocyte membrane to the membrane skeleton and contains 24 ANK repeats that form a spiral domain. Ankyrin-R stabilizes the erythrocyte membrane and mutations in ANK repeats are documented in hereditary spherocytosis (HS), the life-threatening human anemia. We recently examined the mechanical properties of 24 ankyrin-B repeats with atomic force microscopy (AFM) and found that they behave as extremely strong and resilient nanosprings. However, nothing is presently known about molecular mechanisms underlying the nanomechanical properties of wild type ankyrin repeats and their mutants. The long term goal of this application is to elucidate the molecular mechanisms governing the mechanical properties of ANK repeat proteins and to test the hypothesis that the HS-related mutations in ANK repeats of ankyrin-R compromise its nonospring properties, which in turn leads to the conversion of erythrocytes to spherocytes. These objectives can only be achieved by directly testing the mechanical properties of individual ANK repeat proteins and their mutants. Because ankyrins are only ~10 nm in length, the measurements of their elasticity are challenging and require the use of nanotechnology tools such as AFM that allows manipulating single molecules under nearly in vivo conditions. In aim 1, we will combine protein engineering techniques with AFM-based single-molecule force spectroscopy to determine whether the nanospring properties of ankyrin-B, are associated with ankyrin-R and other structurally related ANK repeat proteins. In aim 2, we will use site-directed mutagenesis and AFM to directly evaluate the effects of H277R and V463I mutations in ankyrin-R that cause spherocytosis, on its nanomechanical properties. In aim 3, we will engineer synthetic ANK repeat proteins and a number of ANK repeat mutants, which will be examined by X-ray crystallography, CD spectroscopy, molecular dynamics simulations and AFM in order to identify the amino acids that are responsible for the spiral shape, stability, tensile strength and unfolding/refolding properties of ANK repeat proteins. This research will further our understanding of the relationships between the structure of ANK repeat proteins and their nanomechanics. The study of HS-related ANK repeat mutant proteins may also contribute to an increased understanding of the underlying mechanical cause of spherocytosis, an important human disease. Thus, our project integrates nanoscience and nanotechnology approaches to address important biological and medical problems. Ankyrin (ANK) repeats are identified in thousands of proteins and they play an important role in stabilizing the erythrocyte membrane. Known mutations in ANK repeats are documented in hereditary spherocytosis (HS), the most common, life-threatening inherited hemolytic anemia in humans. This research, which exploits atomic force microscopy for direct measurements of the elastic properties of ankyrin repeats, will further our understanding of the relationship between the structure of these proteins and their spring-like properties.